Compositions and methods for treatment of Gcase related disease states

ABSTRACT

Disclosed are compositions and methods of treating a neurodegenerative disease in an individual. The methods disclose administration of an Integrin α4β1, Very Late Antigen-4 positive neural precursor cell (“VLA4+ NPC”) transfected with a lentivirus overexpressing wild type GCase to an individual having a neurodegenerative disorder. The neurodegenerative disease may include lipid storage diseases, for example Gaucher disease, Parkinson&#39;s disease (PD), Dementia with Lewy bodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of 62/458,628, filedFeb. 14, 2017. The contents of which are incorporated in their entiretyfor all purposes.

BACKGROUND

Lysosomal storage diseases such as Globoid cell leukodystrophy, GM2gangliosidosis, Niemann-Pick C, Mucopolysaccharidoses, Fabry, Tay-Sachs,Sandhoff and Hypercholesterolemia and Gaucher Disease are associatedwith increased cellular immune inflammation and have limited treatmentoptions. Gaucher disease (“GD”), in particular, is a rare disease withan incidence of about 1 in 60,000 in the general population and 1 in 850among Ashkenazi Jewish populations. Worldwide there are about 121,522Gaucher disease patients and here in the US, approximately 5000Americans are suffering from this disease.

GD results from mutations in the glucocerebrosidase gene GBA1 causingfunctional disruption of the encoded lysosomal enzyme, acidbeta-glucosidase, leading to excess accumulation of glucosylceramide(GC) mainly in macrophages (Mϕs) and elevated plasma level of cytokinesand chemokines in human GD patients. Acid beta-glucosidase is crucialfor the degradation of GC into glucose and ceramide. The excessaccumulation of GC in innate and adaptive immune cells within severalvisceral organs, bone and brain sparks a pro-inflammatory environmentresulting in tissue recruitment of several inflammatory immune cells.This pro-inflammatory environment causes tissue damage and promotesclinical GC manifestation.

Improved treatments are needed. Currently, the cost to treat anindividual with enzyme replacement therapy is significant, in the rangeof approximately $100,000 to $300,000 per year. Similarly, substratereduction therapy (e.g., eligustat and miglustat) is equally expensive.While alternative treatments have potential, such as gene therapy,substrate reduction therapy, and alternative enzyme replacementproducts, such treatments have been hampered by limitations in theunderstanding of disease pathogenesis and toxicity concerns due to theblood brain barrier and procedural risks (particularly with respect togene therapy methods).

Thus, there is an urgent need for alternative therapeutic options forthe above-noted disease states and disease states of similar etiology.Further alternative treatments are needed for the management of diseasecomplications in GD and other lysosomal storage diseases associated withincreased cellular immune inflammation. The instant disclosure satisfiesone or more of these needs in the art.

BRIEF SUMMARY

Disclosed are compositions and methods of treating a neurodegenerativedisease in an individual. The methods disclose administration of anIntegrin α4β1, Very Late Antigen-4 positive neural precursor cell(“VLA4+ NPC”) transfected with a lentivirus overexpressing wild typeGCase to an individual having a neurodegenerative disorder. Theneurodegenerative disease may include lipid storage diseases, forexample Gaucher disease, Parkinson's disease (PD), Dementia with Lewybodies.

BRIEF DESCRIPTION OF THE DRAWINGS

This application file contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee. Those of skill in the art will understand that thedrawings, described below, are for illustrative purposes only. Thedrawings are not intended to limit the scope of the present teachings inany way.

FIG. 1A-1E. Enrichment and characterization of VLA4+mNPCs. (FIG. 1A) WTGFP+mouse (m) iPSC-derived NPCs had 11% VLA4+ cells stained withanti-mouse VLA4 antibody. All mouse NPCs are GFP positive. DAPI stainscell nuclei. (FIG. 1B) FACS using anti-VLA4 antibody enriched VLA4+GFP+mNPCs. (FIG. 1C) The FACS sorted cells were stained by anti-VLA4antibody. Scale bar=xx nm. (FIG. 1D) VLA4+m NPCs are validated by neuralstem cell markers, anti-Nestin and anti-SOX2 antibodies. (FIG. 1E)VLA4+mNPCs were differentiated into neurons, oligodendrocytes, andastrocytes. DAPI stains cell nuclei. Scale bar=xx nm.

FIG. 2A-2F. The GFP+VLA4+mNPCs engrafted into the brains of 4L;C* miceby intravenous administration. GFP+VLA4+mNPCs (1×10⁶ cells) weretransplanted by intravenous injection, twice per week, into 4L;C* micestarting at 30 days of age. (FIG. 2A) Donor GFP+mNPCs were detected inthe VCAM1+ endothelia cell layer in the brain, particularly in thebrainstem, midbrain and thalamus. Representative images of some donorGFP+ cells acquiring (FIG. 2B) glial phenotype that are positive forGFAP, (FIG. 2C) neuronal phenotype that are positive for NeuN), and(FIG. 2D) oligodendrocyte phenotype that are positive for 04. (FIG. 2E)qRT-PCR data showed number of GFP positive cells in midbrain. (FIG. 2F)The distribution of GFP positive cells in the mouse brain with 2×IV/week injections).

FIG. 3A-3C. Transplantation of GFP+VLA4+mNPCs (mNPCs) into 4L;C* miceprolonged survival and improved sensorimotor function. (FIG. 3A) mNPCstransplanted 4L;C* mice had significantly (p<0.05) prolonged survivalcompared with Vehicle (saline) injected 4L;C* mice with treatmentregime, one IV/week, two IVs/week or three IVs/week. (FIG. 3B) Thehindpaw clasping test, a marker of neurodegenerative disease, showedsignificantly delayed clasping in the mNPC transplanted 4L;C* mice (twoIVs/week and three IVs/week) compared to saline-4L;C* control. (FIG. 3C)Transplantation of mNPCs significantly improved sensorimotor function in4L;C* mice at 50 days of age by gait analysis for the mice withtreatment of one IV/week, two IVs/week and three IVs/week. Student'st-test.

FIG. 4A-4D. Reduced CNS-inflammation in mNPC transplanted 4L;C*. Thesections from 50-days-4L;C mice were stained with inflammation markers,anti-CD68 antibody (brown) (FIG. 4A) for activatedmicroglial/macrophages and anti-GFAP (brown) (FIG. 4B) antibody forastrogliosis. Quantitative data showed significantly reduced CD68 (FIG.4C) and GFAP (FIG. 4D) signals in brainstem, midbrain and thalamus oftreated 4L;C mice compared to vehicle-4L;C* control, indicatingdecreased inflammation. Student's t-test. *, p<0.05, **, p<0.01.

FIG. 5. mNPC transplantation mitigated neurodegeneration. Fluoro-JadeC-(FJC-) positive cells were examined in the mice brains at 50 days ofage (A) and about 60 days of age at terminal stage in 4L;C* mice treatedwith mNPC or saline. FJC-positive signals were reduced in midbrain,brainstem and thalamus regions. Quantitative data showed significantlyreduction of neurodegeneration (FJC signals) at 50 days of age and atterminate stage in mNPC treated 4L;C* brains compared to vehicle-4L;C*mice. Student's t-test. *, p<0.05. (B)

FIG. 6A-6F. Analyses of GCase and substrate levels in mNPC treatedbrain. (FIG. 6A-FIG. 6D) GCase activity. GCase activity in midbrain ofmNPC treated 4L;C* mice was significantly increased by 35% at 50 days ofage (p=0.002) (FIG. 6A) and modestly increased by 16% at end-stage (FIG.6B) compared to that in vehicle-4L;C* mice. In the brainstem, mNPCtreatment enhanced the GCase activity by 28% (C) at the 50 days of ageand by 16% end-stages (FIG. 6D) compared to that in vehicle-4L;C* mice.The data represent the mean±S.E. (n=3 mice), assayed in triplicate.Student's t-test. ***, p<0.001. (E and F) Substrate levels. GC levels(FIG. 6E) and GS levels (FIG. 6F) in the midbrain of mNPC transplanted4L;C* mice at 50 days of age showed 34% and 23% reduction compared tothat in vehicle-4L;C* mice. The data represent the mean±S.E. andanalyzed by Student's t-test. *, p<0.05 (n=3 mice). The GC and GS levelswere normalized by mg wet tissues.

FIG. 7 Improved mitochondrial function in mNPC transplanted 4L;C* micebrain. Vehicle-4L;C* brain had decreased mitochondrial function with 42%of ATP production, 30% of maximal respiration, 21% of residualrespiration OCR rate compared to WT brains. mNPC treatment improved4L;C* brain ATP production, maximal respiration, residual respirationOCR rate to 64%, 66%, 64% of WT level, respectively. One-way ANOVA withpost-hoc Tukey test (P<0.05), n=3 mice/group, 6 replicates/sample/assay,duplicate assays.

FIG. 8A-8D. The mRNA expression of neuron trophic factors in 4L;C* micebrain. The mRNA expression of BDNF (FIG. 8A), NT-3 (FIG. 8C) and GDNF(FIG. 8E) in 4L;C* brain were determined by qRT-PCR. BDGF, NT-3 and GDNFmRNA levels were increased at 1 mons after NPCs injection and slightlywere decreased after four weeks. The mRNA expression of BDNF (FIG. 8B),NT-3 (FIG. 8D) and GDNF (FIG. 8F) in cells were consistent with theresults.

FIG. 9A-9B. Isolation of VLA4+ human and mouse iPSC-derived NPCs andmouse C17.2 NPCs. A portion of human and mouse iPSC-derived NPCs andmouse C17.2 NPCs express VLA4. (FIG. 9A) Fluorescence-activated cellsorting of VLA4+ NPCs by flow cytometry (FACS). (FIG. 9B) Human andmouse VLA4+ NPCs maintain NPC morphology.

FIG. 10. VLA4+ human GD2 iPSC-derived NPCs maintain NPC properties andmultipotency. Compared to total NPCs, VLA4+ NPCs are stained positivefor neural stem cell markers, anti-Nestin and anti-Sox2 (A), and havepotency to differentiate into neurons (Tuj1, red), oligodendrocytes (04,red), and astrocytes (GFAP, red) (B). DAPI stains cell nuclei.

FIG. 11. Characterization of C17.2 cells. C17.2 cells have neural stemcells properties stained positive by anti-Nestin and anti-Sox2antibodies (A) and have potency (B) to differentiate into neurons (Tuj1,red), oligodendrocytes (04, red), and astrocytes (GFAP, red). DAPIstains cell nuclei.

FIG. 12. Characterize VLA4+C17.2 cells. VLA4+C17.2 cells were isolatedby FACS. (A) The FACS sorted cells were stained positive by anti-VLA4antibody. VLA4+C17.2 cells were validated by neural stem cell markers,anti-Nestin and anti-SOX2 antibodies. (B) VLA4+C17.2 cells weredifferentiated into neurons (Tuj1, red), oligodendrocytes (04, red), andastrocytes (GFAP, red). DAPI stains cell nuclei.

FIG. 13. Engraftment of VLA4+C17.2 NPC in GD mouse brains. (A) GFPlabeled VLA4+C17.2 cells engrafted into GD mouse brains by IVadministration. The mouse brains were collected 2 days, 3 days or 7 dayspost last injection. The VLA4+ cells detected by anti-VLA4 or anti-GFPantibodies were detected in mice brains received IV delivered VLA4+C17.2NPCs. None of VLA4+ cells were detected in PBS injected mice brain. (B)The GFP positive C17.2 NPCs were counted in sagittal brain sections oftreated mouse brain, 10 images per mouse.

FIG. 14. Effect of transplantation of mNPCs on sensorimotor function.Gait analysis of 4L;C* mice at 40 days of age (A) and terminate stage(B). 4L;C* mice were administered mNPCs by one IV/week, two IV/week andthree IV/week. Student's t-test.

FIG. 15. Analysis of liver and brain from transplanted mice. (A) GCaseactivity in the liver was not altered in the mNPC treated 4L;C* micecompared to vehicle control at 50 days of age. (B) Substrate analysis interminal age mice showed that GC and GS levels in NPC treated 4L;C*brain were not significant different from vehicle-4L;C*.

FIG. 16A-16F. Neurotrophic factor expression. The mRNA expression ofneurotrophic factors in mice brains and cells were determined byqRT-PCR. Compared to vehicle-4L;C* brain, CNTF (FIG. 16A) and TNF (FIG.16B) expressed in NPC showed decreased expression in treated 4L:C*brain; IGF1 (FIG. 16C) and GFG2 (FIG. 16D) did not show superiorexpression in NPC over fibroblasts, and their levels were decreased intreated 4L;C* brain; JAG1 (FIG. 16E) and TGFb2 (FIG. 16F) did notexpress in NPC, their levels were increased in treated 4L;C* brain.(Duplicated experiments with triplicated sample/experiment, n=3 mice).

FIG. 17. Scheme: IV delivered VLA4+ NPC cross BBB through interactionwith VCAM1. The therapeutic efficacy was achieved by functional cellreplacement, providing neurotropic function and normal GCase, protectingmitochondrial function, suppressing CNS inflammation neurodegeneration.

FIG. 18. Approach of genetic correction of GBA1 mutation for celltherapy. The single stranded guide (g) RNAs are designed to target themutation, L444P (GBA1 mutation at nt14446, T>C) on iPSCs. The correctediPSC will be derived to neural precursor cells (NPC) for celltransplantation.

FIG. 19. gRNA design for genetic correction of GBA1 mutation L444P.Sequences of gRNA targeting the mutation L444P (GBA1 mutation atnt14446, T>C), a phosphorothioate-modified single strandedoligonucleotide (ssODN) donor template and genotyping primers.

FIG. 20. Sequence of corrected clone 9. An example of the correctedclone #9 was verified for the correction of the mutant allele and notaffect WT allele by Sanger sequencing.

FIG. 21. Restored GCase activity in the genetically-corrected iPSCclones. Corrected GD-iPSC clones are assayed for GBA1 encoding enzymeacid β-glucosidase (GCase) activity to confirm restoring GCase incorrected clones. Corrected clones (#9, #27, #44) recovered GCaseactivity at ˜40-47% of normal level, compared to the control H1 clonewith normal GBA1 gene, which reached to the heterozygous GBA1 levels.

FIG. 22A-22B. Generation of human GBA1 expression lentiviral vector.Human WT GBA1 cDNA was cloned into the two lentiviral vectors (shown in22A and 22B) using In-Fusion Cloning kit. The lentiviral vectors werepackaged into viral particles. 1 and 2 showing cloning sites for eachvector.

FIG. 23. IF detection of lentiviral vector marker (mCherry or GFP) inGFP+VLA4+ NPCs transduced with human GBA1 expression lentiviralparticles.)

FIG. 24. GCase activity assay determined that human GBA1 was expressedand secreted in the NPCs transduced by lentiviral vectorPLVX-GBA1-mCHERRY that was used in following cell and mice studies.Lentiviral vector FUGW-GBA1-GFP transduced cells did not havesignificant GCase secretion and was not used for following studies.

DETAILED DESCRIPTION

Definitions

Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art. Incase of conflict, the present document, including definitions, willcontrol. Preferred methods and materials are described below, althoughmethods and materials similar or equivalent to those described hereincan be used in practice or testing of the present invention. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. Thematerials, methods, and examples disclosed herein are illustrative onlyand not intended to be limiting.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and reference to “a dose” includes referenceto one or more doses and equivalents thereof known to those skilled inthe art, and so forth.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to an animal that is the object of treatment,observation and/or experiment. Generally, the term refers to a humanpatient, but the methods and compositions may be equally applicable tonon-human subjects such as other mammals. In some embodiments, the termsrefer to humans. In further embodiments, the terms may refer tochildren.

There is no effective treatment available for neuronopathic Gaucherdisease characterized by progressive neurodegeneration phenotype. Atherapy of non-invasive transplantation of neural precursor cells fordisease states such as neuronopathic Gaucher disease is disclosed.

Gaucher disease is caused by GBA1 mutations leading to functionaldeficiency of lysosomal acid-β-glucosidase. GBA1 mutations that causeGaucher disease have been identified as the most common genetic riskfactor for Parkinson's disease. Applicant has developed a non-invasive,autologous induced pluripotent stem cell (iPSC)-based cell and genetherapy for treatment of diseases such as neuronopathic Gaucher diseaseand Parkinson's disease. A subclass of neural stem and precursor cells(NPCs), including iPSCs-derived NPCs, express VLA4 (Integrin α4β1, VeryLate Antigen-4) which allows NPCs to cross the blood-brain-barrier.

Applicant has found that the GBA1 mutation in human Gaucher diseaseiPSCs can be genetically corrected and iPSC-derived NPCs having a VLA4+population can be established. Applicant has found that the VLA4+ NPCsmaintained multipotency and differentiated into neurons, astrocytes, andoligodendrocytes. The non-invasive delivery of iPSC derived VLA4+ NPCsbenefit individuals having Parkinson's disease by increasing GBA1 geneproduct, promoting neuroprotection and direct cell replacement throughdifferentiation to functional cell types in the central nervous system.

A subclass of neural stem and precursor cells (NPCs), including inducedpluripotent stem cell (iPSC)-derived NPCs, express VLA4 (Integrinalpha4beta1, Very Late Antigen-4) allows systemically delivered NPCs tocross the blood-brain-barrier via interaction with endothelial VCAM 1(vascular cell adhesion molecule). Applicant has established multipotenthuman and mouse iPSC-derived VLA4+ NPCs. Mouse iPSC-derived VLA4+ NPCsexpressing GFP engrafted in neuronopathic Gaucher disease (4L;C*) mousebrain following intravenous injection. GFP positive cells weredistributed in brain stem, midbrain, and thalamus in the transplanted4L;C* brains and were differentiated into neurons, astrocytes andoligodendrocytes. The NPC transplanted 4L;C* mice showed improvedsensorimotor function by gait analysis and delayed neurodegenerativedisease progression by the hindlimb clasping test. NPC transplantationsignificantly prolonged life span of 4L;C* mice. Histological analysisof brain sections by Fluoro-Jade C staining showed significantly reducedneurodegeneration in the brain stem, midbrain, and thalamus oftransplanted mice. CNS inflammation, detected by anti-CD68 and anti-GFAPantibodies, was significantly decreased in the brain and spinal cord oftransplanted mice. The oxygen consumption rate of NPC treated brainmitochondria was significantly improved compared to vehicle-mice. Theseresults demonstrated the potential efficacy of non-invasive delivery ofiPSC-derived NPCs to improve neuropathic phenotype in the mouse model ofneuronopathic Gaucher disease. Applicant has thus established thefeasibility of non-invasive, autologous cell therapies usingiPSC-derived precursor cells to achieve personalized medicine forneuronopathic Gaucher disease and other neurodegenerative diseases.

In one aspect, a method of treating a neurodegenerative disease in anindividual in need thereof is disclosed. The method may, in someaspects, comprise the step of administering to the individual anIntegrin α4β1, Very Late Antigen-4 positive neural precursor cell(“VLA4+ NPC”) The VLA4+ NPC may be transfected with a lentivirusoverexpressing wild type GCase. The neurodegenerative disease may beselected from Gaucher disease, Parkinson's disease (PD), or Dementiawith Lewy bodies. In one aspect, the neurodegenerative disease may beone which is characterized by reduced GCase activity, accumulation ofsubstrates (GC), and/or alpha synuclein aggregation. In one aspect, theneurodegenerative disease may be one which is caused or exacerbated by aGBA1 mutation. In one aspect, the Gaucher disease may be type II nGD. Inone aspect, the Gaucher disease may be type III nGD.

In one aspect, the NPC may be derived from an iPSC. The iPSC may bederived from a human fibroblast. Human iPSCs as described herein wereestablished in Pluripotent Stem Cell Core at CCHMC from healthindividual or GD patients' fibroblasts, using methods known in the art.An exemplary protocol and study were published in Sun, Y., et al. (2015)PLoS One 10(3): e0118771. Mouse iPSCs may be derived from wild typeGFP+mouse fibroblasts.

In one aspect, the VLA4+ NPC may be administered to said individual viaintravenous administration. One of ordinary skill in the art willreadily appreciate suitable carriers for IV administration, such as, forexample, sterile solutions commonly used in the art.

In one aspect, the lentivirus may comprise a promoter capable of drivingtransgene expression in the central nervous system. For example, thepromoter may be selected from a human elongation factor 1 alpha (“EF1α”)promoter or an Ubiquitin C promoter (UbC). Other suitable promoters willbe readily understood by one of ordinary skill in the art.

In one aspect, the VLA4+ NPC may be co-administered with a chaperonemolecule. For example, the VLA4+ NPC may be co-administered with achaperone molecule such as Dantrolene, Ambroxol, or a combinationthereof. Dantrolene and Ambroxol can access a variety of organsincluding the CNS to restore mutant GCase activity acting aschaperon-inducer or chaperone. Dantrolene, an antagonist of ryanodinereceptors, modulates ER-calcium mobilization to regulatecalcium-dependent chaperones for GCase protein folding to enhance itsfunction. Dantrolene has been used to safely to treat malignanthyperthermia. Applicant has shown that Dantrolene is capable ofrepairing mutant GCase activity in GD mutant fibroblasts and nGD miceproviding a therapeutic option for GD. Ambroxol, a commonly usedexpectorant, has been in pilot studies to evaluate its safety andefficacy in vGD and nGD patients and in a Phase I trial for vGD(ClinicalTrials.gov Identifier: NCT01463215) and may be used with thedisclosed methods. Ambroxol binds to GCase in a pH dependent manner,assisting in folding at the neutral pH of the ER and releases from GCaseat the acidic pH of the lysosome, and may further be used with thedisclosed methods.

In one aspect, the VLA4+ NPC may be delivered in an amount sufficient toincrease GCase activity in the brain. In one aspect, the VLA4+ NPC maybe delivered in an amount sufficient to improve one or more parametersselected from neurological pathology, survival, brain inflammation,brain neurodegeneration, GCase activity, GCase substrate level,mitochondrial function, neurotropic factor expression, or combinationsthereof.

In one aspect, the VLA4+ NPC may be co-adminstered with an ERT(imiglucerase, velaglucerase alfa, taliglucerase alpha) or SRT(eligustat, miglustat, ibiglustat/Verglustat, or combinations thereof,preferably ibiglustat/Verglustat.

In one aspect, a method of treating a neurodegenerative disease in anindividual having one or more mutations in a GCase gene is disclosed.The method may comprise the step of administering to the individual anIntegrin α4β1, Very Late Antigen-4 positive neural precursor cell(“VLA4+ NPC”), wherein fibroblasts or other somatic cells for generatingiPSC and the VLA4+ NPC may be harvested from the individual, and whereinthe VLA4+ NPC may contain one or more corrected GCase genes. In oneaspect, the corrected GCase gene may be a CRISPR-corrected GCase gene,using methods known in the art. (See, e.g., Ran F A, Hsu P D, Wright J,Agarwala V, Scott D A, et al. (2013) Genome engineering using theCRISPR-Cas9 system. Nature protocols 8: 2281-2308.) In one aspect, theVLA4+ NPC containing one or more CRISPR-corrected GCase genes may beadministered intravenously to an individual in need thereof.

In one aspect, one or more CRISPR corrected genes may be achieved bycarrying out one or more of the following steps: contacting an iPSCderived from said individual with a guide RNA (“gRNA”) specific for atargeted genomic region containing one or more mutations and aCRISPR-associated endonuclease (examples include, but are not limitedto, Cas9, S. pyogenes Cas9 (SpCas9), Cpf1, or the like) in an amount andfor a duration sufficient to convert said mutation to a wild-typesequence; assessing CRISPR-Cas9 cleavage activity; genotyping editedhuman iPSC cell clones; functionally screening said edited human iPSCcell clones via an enzyme activity assay; karyotyping for chromosomalanalysis of the edited human iPSC cell clones; differentiating said iPSCcells to neural precursor cells (NPCs); deriving a VLA4+ NPC enrichedpopulation from said NPCs, preferably wherein said step of deriving saidVLA4+ NPC population is carried out by subjecting the NPCs to a FACSsorting step.

In one aspect, the VLA4+ NPC enriched population may comprise at least50% NPC cells that are VLA4+, or at least 60% NPC cells that are VLA4+,or at least 70% NPC cells that are VLA4+, or at least 80% NPC cells thatare VLA4+, or at least 90% NPC cells that are VLA4+, or about 100% NPCcells that are VLA4+. The VLA4+ NPC enriched population may beadministered to an individual in an amount of at least about 1×10⁶ cellsper injection. In one aspect, the VLA4+ NPC enriched population may beadministered at a concentration of about 1×10⁶ cells/100 ul. In oneaspect, the VLA4+ NPC enriched population may be administered in acarrier selected from saline or phosphate buffered saline. In oneaspect, the VLA4+ NPC enriched population may be co-administered with aneurotropic factor in an amount sufficient to increase cell engraftment.In one aspect, the VLA4+ NPC population may be administered 1 to 3 timesper week. In certain aspects, the NPC cell clearance may be monitored inblood by Flow cytometry using neural stem cell marker (e.g nestin). Inone aspect, the VLA4+ NPC may further comprise a detectable tag, forexample, GFP, by transduction of lentiviral GFP into NPCs. In certainembodiments, the GFP tag can be used to track the tissue distribution ofengrafted NPCs.

In one aspect, the targeted genomic region may be a region wherein afunctional mutation is located. For example, there are more than 400known GBA1 mutations. (See, e.g., Grabowski G A, Zimran A, Ida H.“Gaucher disease types 1 and 3: Phenotypic characterization of largepopulations from the ICGG Gaucher Registry”. Am J Hematol. 2015 July; 90Suppl 1:S12-8. doi: 10.1002/ajh.24063. Review) In one aspect, the VLA4+NPC population may be assayed for secretion of wild-type GCase proteinprior to administration to an individual in need thereof.

In one aspect, a method of transplanting a cell in the central nervoussystem of an individual is disclosed. In this aspect, the method maycomprise administering a VLA4+ NPC derived from the individual, whereinthe administration may be intravenous. The method may be used to treat aneurodegenerative disease such as Gaucher disease, Parkinson's disease(PD), Dementia with Lewy Bodies, or a lysosomal disease with a brainmanifestation, including lysosomal diseases with brain diseases, or alysosomal disease with a brain manifestation, including lysosomaldiseases with brain diseases. The neurodegenerative disease may be onecharacterized by reduced GCase activity and/or alpha synucleinaggregation, for example, neuronopathic Gaucher disease, or aneurodegenerative disease caused by a GBA1 mutation. In certain aspects,the NPC may be derived from an iPSC. As set forth above, the VLA4+ NPCmay be co-administered with a chaperone molecule as described. The VLA4+NPC may be delivered in an amount sufficient to improve one or moreparameters selected from neurological pathology, survival, braininflammation, brain neurodegeneration, GCase activity, GCase substratelevel, mitochondrial function, neurotropic factor expression, orcombinations thereof in the treated individual.

In one aspect, a medicament for the treatment of a neurodegenerativedisease comprising an Integrin α4β1, Very Late Antigen-4 positive neuralprecursor cell (“VLA4+ NPC”) transfected with a lentivirusoverexpressing wild type GCase, as described in any of the embodimentsabove, is disclosed.

EXAMPLES

The following non-limiting examples are provided to further illustrateembodiments of the invention disclosed herein. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples that follow represent approaches that have been found tofunction well in the practice of the invention, and thus can beconsidered to constitute examples of modes for its practice. Those ofskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments that aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the invention.

Applicant has genetically corrected GBA1 mutation in human iPSCs derivedfrom an individual with Gaucher disease (GD). Specifically, CRISPR/Cas9was used to correct the L444P mutation in an iPSC line harboringcompound heterozygous GBA1 mutations (L444P/P415R).

In the following examples, patient iPSCs are from GD neuronopathicvariants having GBA1 mutations: L444P/P415R. L444P is second mostprevalent GD causing mutation. It is a severe mutation that, inhomozygosity or compound heterozygosity with other mutations, leads toneuropathic type GD.

The clone originally selected for correction (iPSC47_32) was onedescribed in Sun, Y., et al. (2015) PLoS One 10(3): e0118771, whichdescribes the generation of GD-specific human iPSCs. Applicant hasgenerated iPSCs from fibroblasts derived from both human GD patients andnormal individuals. The iPSCs can differentiate into NPCs using aroutine neural aggregation-based protocol. (Sun, Y., et al. (2015) PLoSOne 10(3): e0118771). NPCs can be stably passaged in vitro whilstmaintaining the expression of nestin and sox2. NPCs possess multipotencywhen they were cultured with FGF8 and SHH. Resulting cultures containedboth GFAP+ astrocytes and Tuj1+ neurons, demonstrating multipotency.Neurons possessed prototypical electrophysiological properties whenassayed by whole cell patch-clamp. (Sun, Y., et al. (2015) PLoS One10(3): e0118771).

The single stranded guide (g) RNAs are designed to target the mutation,L444P (GBA1 mutation at nt14446, T>C) on iPSCs (See, e.g., FIGS. 1 and2). The corrected clones are verified for the correction of the mutantallele and no effect on WT allele by DNA sequencing (FIG. 3). Theconstruction of gRNA, validation of gRNA editing activity and DNAsequence in iPSCs are carried out in CCHMC Transgenic Animal & GenomicEditing Core using methods known in the art. The corrected iPSC clonescarry heterozygous GBA1 mutations P415R (wt/P415R). Because GD isautosomal recessive disease, people with heterozygous GBA1 mutation,like GD carrier, have sufficient GCase function to live normal life.

Corrected GD-iPSC clones are assayed for GBA1 encoding enzyme acid(3-glucosidase (GCase) activity to confirm restoring GCase in correctedclones. Corrected clones (herein referred to as #9, #27, #44) recoveredGCase activity at ˜40-47% of normal level, compared to the control H1clone with normal GBA1 gene (FIG. 4), which reached heterozygous GBA1levels.

Corrected GD-iPSC can be validated for chromosome normality byKaryotyping. The edited iPSCs can be derived to NPCs and FACS sorted forVLA4+ population. The corrected VLA4+ NPCs can then be tested formultipotency. In vivo efficacy of corrected VLA4+ NPCs can then beevaluated in animal models by intravenous injection, a noninvasiveprocedure, to assess the ability of the corrected cells to engraft inthe mouse CNS and provide short-term amelioration of substrateaccumulation and inflammatory phenotypes in the GD mouse brain(transplanted human NPCs survive only 1-2 months in the CNS ofimmunocompetent mice). In parallel, uncorrected GD and normal humaniPSC-derived NPCs can be analyzed.

Genetically edited GD-iPSCs may be used as a personalized therapy byautologous cell replacement (NPCs and other cell types) treatments forGD patients, with wider applications in Parkinson's disease.

Methods

Generation of Human GBA1 Expression Lentiviral Vector.

Human wild-type GBA1 cDNA is from Origene™ Technologies (GBA(untagged)-Human glucosidase, beta, acid (GBA), transcript variant 3, NM001005742.1). The full-length GBA cDNA is cloned into flap-Ubpromoter-GFP-WRE lentiviral expression vector (Addgene, Plasmid #14883)and pLVX-EF1α-IRES-mCherry Vector (Clontech, Catalog No. 631987) usingIn-Fusion Cloning kit (Clontech). The GBA1 clones are confirmed by DNAsequencing. The lent vector constructs are packaged into viral particlesusing HEK293T cells and lentiviruses are produced at CincinnatiChildren's Hospital Medical Center vector core using methods known inthe art. The titer of pLVX-EF1α-GBA1-IRES-mCherry andFUGW-GBA1-IRES-EGFP lenti-virus are 8.13 E+07 and 3.51 E+06,respectively.

Transduction of NPCs with Lentiviral hGBA1.

WT VLA4+ GFP+ NPCs are plated at 7×10⁴ cells/well in 6-well plate. Thecells are transduced with Lentivector PLVX-GBA1-IRES-mCherry (MOI=23)and FUGW-GBA1-IRES-EGFP (MOI=1), respectively, in serum-free medium inthe presence of Polybrene (4 μg/ml) (an enhancer reagent ofretrovirus-mediated gene transduction). Transduced cells are sorted byFACS for GFP or mCherry-positive cells that contained FUGW-GBA1-GFP orpLVX-EF1α-GBA1-IRES-mCherry. The transduced NPCs are fixed by 4% PFA andevaluated for GFP or mCherry signals by Phase contrast andimmunofluorescence using conventional fluorescence microscopy (ZeissAxiophot; Oberkochen, Germany).

GCase Activity Assay.

The cells are homogenized in 1% Na-taurocholate/1% Triton X-100 (Tc/Tx).Mouse tissues are homogenized in 1% Tc/Tx. GCase activity is determinedfluorometrically with 4MU-Glc as substrate in 0.25% Tc/Tx diluted in0.1M citrate phosphate (CP) buffer (pH 5.6) as described. Liou, B. etal. Analyses of variant acid beta-glucosidases: effects of Gaucherdisease mutations. J Biol Chem 281, 4242-53 (2006). Mouse brains arehomogenized in 1×PBS and incubated in 5 mM brain phosphatidylserine. TheGCase activity assay is carried out in 0.1M CP buffer (pH 5.6) using4MU-Glc as substrate as described. (Xu, Y. H. et al. Dependence ofreversibility and progression of mouse neuronopathic Gaucher disease onacid beta-glucosidase residual activity levels. Mol Genet Metab 94,190-203 (2008).) Protein concentrations of cells and tissues weredetermined by BCA assay using BSA as standard.

Genomic Editing Patient iPSC Clones.

The iPSCs are derived from both human GD patients and normal individualsas described in (Sun, Y., et al. (2015) PLoS One 10(3): e0118771). Theclone selected for correction is iPSC47_36 that carries compoundheterozygous GBA1 mutation (P415R/L444P). The single stranded guide (g)RNA was designed to target spCas9-mediated double strand breakintroduction proximal to the L444P mutation (GBA1 mutation at nt14446,T>C) in iPSC47_36. Oligonucleotides (caccGAAGAACGACCcGGACGCAG (SEQ IDNO:29) and aaacCTGCGTCCgGGTCGTTCTTC) (SEQ ID NO:30) encoding the sgRNAtarget sequence are annealed and subcloned via BbsI restriction digestinto plasmid px458M that contains a U6 promoter-driven sgRNA and aSpCas9 expression cassette. The pX458M plasmid is modified from thepX458 plasmid (addgene #48138) (Ran F A, Hsu P D, Wright J, Agarwala V,Scott D A, et al. (2013) Genome engineering using the CRISPR-Cas9system. Nature protocols 8: 2281-2308) and carries an optimized sgRNAscaffold (Chen B, Gilbert L A, Cimini B A, Schnitzbauer J, Zhang W, etal. (2013) Dynamic imaging of genomic loci in living human cells by anoptimized CRISPR/Cas system Cell 155: 1479-1491.). The targetingactivity of this plasmid is validated by T7E1 assay using 293 cells.iPSC47_36 cells are transfected with pX458M and aphosphorothioate-modified single stranded oligonucleotide (ssODN) donortemplate using TransIT-LT1 (Mirus). The ssODN (FIG. 2) is designed tointroduce the desired wildtype GBA1 sequence flanked by homology arms tothe targeted genomic region. The ssODN is also designed to containsilent mutations to prevent retargeting by spCas9 and to introduce aBtgI restriction site to facilitate identification of targeted clones.Forty-eight hours post-transfection, GFP-positive cells are isolated byFACS and re-plated at cloning density (250-500 cells/well of a 6 wellMatrigel-coated plate). Cells are re-plated and cultured for 4 days inmTeSR1 containing 10% CloneR (StemCell Technologies), using themanufacturer's recommended protocol. Cells were subsequently fed dailywith mTeSR1 for an additional 9 days before colonies were manuallyharvested and expanded for genotyping. Primers VS4247(gtgcgtaactttgtcgacagtcc) (SEQ ID NO:31) and VS4249(ctgagagtgtgatcctgccaag) (SEQ ID NO:32) are used to PCR amplify thetargeted GBA1 genomic region and products are subjected to BtgIdigestion to identify putative edited clones. Corrected clones aresubsequently confirmed by Sanger sequencing. The construction of gRNA,validation of gRNA editing activity and DNA sequencing are carried outby the CCHMC Transgenic Animal & Genome Editing Core using methods knownin the art.

FACS Isolation of VLA+ NPCs and Testing NPCs Multipotency.

GFP+ mouse iPSC-derived neural precursor cells (NPC) were produced asdescribed (Sun, Y., et al. (2015) PLoS One 10(3): e0118771). The mouseNPCs are cultured in STEMdiff™ Neural Progenitor Medium (STEMCELLTechnologies) and 1×10⁷ mNPCs were labelled by anti-VLA4 antibodyconjugated with APC (1:50, Miltenyi Biotec, 130-102-142) in NPC culturemedium for 30 min. The labeled cells are sorted by FACS in PBS. Thesorted VLA4+ cells are confirmed by staining with anti-VLA4 antibody.For neural differentiation, the VLA4+ NPC are plated on a polyomithineand laminin-coated culture dish in complete StemPro® NSC SFM at2.5-5×10⁴ cells/cm² for 2 days and medium is changed, allowingdifferentiation: neuron differentiation medium (Neurobasal® Medium,B-27® Serum-Free Supplement, GlutaMAX™-I Supplement), aastrocytedifferentiation medium (D-MEM with N-2, GlutaMAX™-I, and FBS),oligodendrocyte differentiation medium (Neurobasal® medium with B-27®,GlutaMAX™-I, and T3). The media is refreshed every 3-4 days (Sun, Y. etal. Properties of neurons derived from induced pluripotent stem cells ofGaucher disease type 2 patient fibroblasts: potential role inneuropathology. PLoS One 10, e0118771 (2015)).

Mice Treatment and Assessment of Survival and Body Weight.

To minimize mixed background interference with behavioral testing,Applicant has generated a C57BL/6 strain of 4L;C* mice by back-crossingto C57BL/6 WT mice for 10 generations. The C57BL/6 4L;C* mice developedthe same neuronal phenotype with an average life span of 56 dayscompared to −48 days for mixed background (C57BL/6J/129SvEV) 4L;C* mice(Sun, Y. et al. Neuronopathic Gaucher disease in the mouse: viablecombined selective saposin C deficiency and mutant glucocerebrosidase(V394L) mice with glucosylsphingosine and glucosylceramide accumulationand progressive neurological deficits. Hum Mol Genet 19, 1088-97(2010).). These C57BL/6 4L;C* mice have a sufficient lifespan to allowIV tail injection and for use in this study. 4L;C* mice are transplantedwith the NPCs by IV tail injection with 1×10⁶ cells/injection, oneIV/week, 2 IV/week or 3 IV/week, until end stage. 4L;C* mice aremonitored following transplantation for body weight and clinical signsof disease. The mice are euthanized by sodium pentobarbital at theclinical end point when presenting difficulties in feeding, a cleardownward trend of body weight loss, and severe paralysis, as previouslydescribed (Sun, Y. et al. Neuronopathic Gaucher disease in the mouse:viable combined selective saposin C deficiency and mutantglucocerebrosidase (V394L) mice with glucosylsphingosine andglucosylceramide accumulation and progressive neurological deficits. HumMol Genet 19, 1088-97 (2010).). Mice tissues are collected aftertranscardial perfusion with saline. Dissected tissues were either fixedin 4% paraformaldehyde (PFA) or snap-frozen for further analyses. Theage at end point is analyzed for survival using Prizm software.

Survival and body weight change is shown in Table 1 and Table 2.

TABLE 1 Survival Data mNPC mNPC mNPC Vehicle 1x/WK 2x/WK 3x/WK (Saline)Untreated Range (days) 61-62 60-63 60-64 56-62 51-62 Average (days) 61.561.2 61.4 57.8 56.2 Percentage (vs 106.4 105.9 106.3 100 −2.7 saline) %P value (vs saline) 0.002 0.0202 0.003 0.0912

TABLE 2 Peak body weight data mNPC mNPC mNPC Vehicle 1x/WK 2x/WK 3x/WK(Saline) Age of the peak body 46 47 50 43 weight (days)

Neurobehavioral Testing.

Hindlimb clasping is tested by grasping the tail from base and liftingthe mouse clear of all surrounding objects. The hindlimb position isobserved for 30 seconds. Each mouse at each time point is tested in twotrials, 10 mins apart between two trials, giving a score as follows.Score 0, hindlimbs are consistently splayed outward, away from theabdomen; Score 1, one hindlimb is retracted toward the abdomen for >50%of 30 seconds; score 2, both hindlimbs are partially retracted towardthe abdomen for >50% of 30 seconds; and score 3, hindlimbs are entirelyretracted and touching the abdomen for >50% of 30 seconds. Gait analyseswas conducted to determine sensorimotor function. (Fleming, S. M. et al.Early and progressive sensorimotor anomalies in mice overexpressingwild-type human alpha-synuclein. J Neurosci 24, 9434-40 (2004).) Micewere trained to walk through a narrow alley leading into theirhome-cage. The mice are then brushed with non-toxic paint on the hindpaws and placed at the beginning of the alley. As they walk into theirhome-cage they leave paw prints on the paper underneath. Stride lengthand base width are determined by measuring the distance between hind pawprints. The 4L;C* and control mice, untreated and non-4L;C* littermatesare tested for gait at 40, 50 and over 50 days of age. The investigatorsperforming the functional assessment are blind to the treatment.

Immunofluorescence.

Immunofluorescence staining is performed on PFA fixed brains and cells.The brain sections are incubated in 0.3% Triton X-100 for 30 min, andtreated with 50 mM NH₄Cl in 1×PBS for 15 min followed by 1×PBS wash. Thesections are blocked for 1 hr at RT in Blocking buffer (10% goat serumand 0.4% Triton X-100 in PBS). PFA fixed cells are incubated in blockingbuffer (5% goat serum and 0.4% Triton X-100 in PBS) for 30-60 minutes.Following primary antibodies diluted in 5% goat serum is applied to thecells and brain sections overnight at 4° C. Rabbit anti-GFP (1:200, CellSignaling Technology, 2956), rat anti-CD106 (VCAM-1) monoclonal Antibody(1:100, ebioscience, MR106), mouse anti-SOX2 (1:200, Millipore, AB5603),mouse anti-Nestin (1:100, Millipore, MAB353), mouse anti-integrin alpha4 (VLA4) (1:100, Cell Signaling Technology, 8440S), mouse anti-TUJ1(1:200, STEMCELL TECHNOLOGIES, 60052), mouse anti-04 (1:100, Millipore,MAB365), mouse anti-NeuN antibody (1:500, Millipore, MAB377). Afterwashing in PBS (3×10 min), the secondary antibodies: goat anti-mouseconjugated with Alexa Fluor® 594 (1:1000) for SOX2, Nestin, VLA4, Tuj1,GFAP, and 04 detection, goat anti-rabbit conjugated with Alexa Fluor®488 (1:1000) for GFP detection, and goat anti-rat conjugated with AlexaFluor® 594 (1:1000) for VCAM detection, are applied. The cell andtissues sections are count-stained for nuclei with DAPI in mountingmedium.

Immunohistochemistry. Frozen tissue sections fixed with 4% PFA areincubated with rat anti-mouse CD68 monoclonal antibodies (Serotec,mca1957) (1:3000 in PBS with 5% BSA) and mouse anti-GFAP monoclonalantibody (Roche #7604345). Detection is performed using Research IHC DABXT and Research IHC Omni-UltraMap HRP XT Discovery XT Staining Module.ABC Vectastain and Alkaline Phosphatase Kit II (black) are usedaccording to the manufacturer's instruction. The slides arecounterstained with methylene green. Images are captured using a Zeissmicroscope (Axioskop) equipped with SPOT Advance software (SPOTDiagnostic Instruments, Inc.). CD68-positive and GFAP-positive signal inbrain sections are analyzed by Fiji software in randomly selected fields(300 mm6210 mm/field) from treated and saline control mice brains.Quantification data of the relative positive signal ratio is performed.Data collected from 10 images for each group).

Fluoro-Jade C (FJC) Staining.

FJC is a fluorescent dye derived from fluorescein used to labeldegenerating neurons. (Chidlow, G., Wood, J. P., Sarvestani, G.,Manavis, J. & Casson, R. J. Evaluation of Fluoro-Jade C as a marker ofdegenerating neurons in the rat retina and optic nerve. Exp Eye Res 88,426-37 (2009).) FJC staining and imaging analysis is performed on thefrozen brain sections (25 μm). Prior to staining, sections are mountedon gelatin coated slides that were prepared by immersion in 1% pig skingelatin solution (Sigma; gel strength 300, Type A) and dried overnightin an oven at 60° C. The sections mounted on gelatin slide are air driedfor 30 min in a slide warmer at 50° C. Slides bearing tissue sectionswere first immersed in a solution consisting of 1% sodium hydroxide in80% ethanol for 5 min, followed by rinsing for 2 min in 70% ethanol, 2min in distilled water, and incubating in 0.06% potassium permanganatesolution for 10 min. The tissue slides are then washed for 1-2 min inwater and transferred for 10 min to a 0.0001% solution of FJ C(MILLIPORE, AG325) dissolved in 0.1% acetic acid. The slides are washedthrough three changes of distilled water for 1 min per change and airdried on a slide warmer at 50° C. for 5 min. The air-dried slides aredipped in xylene for 1 min and mounted with DPX (mixture of distyrene, aplasticizer, and xylene) non-fluorescent mounting media (Sigma).

Glycosphingolipids Analysis.

Tissue are homogenized in water and chloroform/methanol using a PowerGen35 (Fisher Scientific) as described. (Sun, Y. et al. Substratecompositional variation with tissue/region and Gba1 mutations in mousemodels—implications for Gaucher disease. PLoS One 8, e57560 (2013).)Aliquots of the homogenates are processed for glycolipids extraction andanalyzed for glucosylceramide and glucosylsphingosine by LC-MS atMedical University of South Carolina Lipidomics SharedResource-Analytical Unit.

Oxygen Consumption Assay.

Mouse brain mitochondria were isolated as described (Liou, B. et al.Modulating ryanodine receptors with dantrolene attenuates neuronopathicphenotype in Gaucher disease mice. Hum Mol Genet 25, 5126-5141 (2016)).To evaluate the mitochondrial function, OCR was measured and the datawere analyzed using the XFe Wave software as described (Liou, B. et al.Modulating ryanodine receptors with dantrolene attenuates neuronopathicphenotype in Gaucher disease mice. Hum Mol Genet 25, 5126-5141 (2016)).ATP production rate in brain mitochondria was normalized to mg ofmitochondrial protein (Dasgupta, N. et al. Neuronopathic Gaucherdisease: dysregulated mRNAs and miRNAs in brain pathogenesis and effectsof pharmacologic chaperone treatment in a mouse model. Hum Mol Genet 24,7031-48 (2015)).

Quantitative Real-Time PCR.

Total RNA from the mouse midbrain is isolated using an RNeasy Micro Kit(QIAGEN) including DNase treatment to remove potential genomic DNAcontamination. The starting RNA (1000 ng) is quantified byspectrophotometric analysis (ND-100; NanoDrop, Thermo Scientific). TotalRNA from mouse midbrain obtained from 3 animals for each condition (WT,NPCs treated and vehicle treated 4L;C* mice) is extracted as described.(Dasgupta, N. et al. Neuronopathic Gaucher disease: dysregulated mRNAsand miRNAs in brain pathogenesis and effects of pharmacologic chaperonetreatment in a mouse model. Hum Mol Genet 24, 7031-48 (2015)). Total RNAis reverse transcribed into complementary DNA using random hexamers andTranscriptor Reverse Transcriptase (Roche Diagnostics). Real-time PCR isperformed according to the manufacturer's protocol using TaqMan GeneExpression Assays and an ABI Prism 7000 Sequence Detection System(Applied Biosystems). The gene expression assays used were thefollowing: Nt3 (Gene ID: 18205; neurotrophin 3), Bdnf (Gene ID: 12064;brain derived neurotrophic factor), Cntf (Gene ID: 12803; ciliaryneurotrophic factor), Tgfb2 (Gene ID: 21808; transforming growth factor,beta 2), Cntfra (Gene ID: 12804; ciliary neurotrophic factor receptor),Igf1 (Gene ID: 16000; insulin-like growth factor 1), Jag1 (Gene ID:16449; jagged 1), Lif (Gene ID: 16878; leukemia inhibitory factor), Tnf(Gene ID: 21926; tumor necrosis factor), Vegfa (Gene ID: 22339; vascularendothelial growth factor A), Vegfb (Gene ID: 22340; vascularendothelial growth factor B), Gdnf (Gene ID: 14573; glial cell linederived neurotrophic factor), Tnfα (Gene ID: 21926; tumor necrosisfactor) and Fgf2 (Gene ID: 14173; fibroblast growth factor 2). Primerssequences are listed in Table 3. The reactions were performed intriplicate and gene expression Ct values were corrected for β-actin Ctvalues using the ΔΔCt method.

TABLE 3 Primers for qRT-PCR Primers (species) Sequence (position)  1NT-3-F-qRT 5′-ATGCAGAACATAAGAGTCAC-3′ SEQ ID NO: 1 NT-3-R-Qrt5′-AGCCTACGAGTTTGTTGTTT-3′ SEQ ID NO: 2  2 BDNF-F-qRT5′-GAAAGTCCCGGTATCCAAAG-3′ SEQ ID NO: 3 BDNF-R-qRT5′-CCAGCCAATTCTCTTTTT-3′ SEQ ID NO: 4  3 CNTF-F-qRT5′-GGCTAGCAAGGAAGATTCGT-3′ SEQ ID NO: 5 CNTF-R-qRT5′-TCCCTTGGAAGGTACGGTAA-3′ SEQ ID NO: 6  4 TGFb2-F-qRT5′-AAAATCGACATGCCGTCCCA-3′ SEQ ID NO: 7 TGFb2-R-qRT5′-ATACCTGCAAATCTCGCCTC-3′ SEQ ID NO: 8  5 CNTFRa-F-qRT5′-ATCCCCAATACCTTCAAT-3′ SEQ ID NO: 9 CNTFRa-R-qRT5′-TATTCCTTCCCTGCGTAG-3′ SEQ ID NO: 10  6 Igf1-F-qRT5′-GCTGGTGGATGCTCTTCAGT-3′ SEQ ID NO: 11 Igf1-R-qRT5′-TAGGGACGGGGACTTCTGAG-3′ SEQ ID NO: 12  7 Jag1-F-qRT5′-GAAAGACCACTGCCGTACCA-3′ SEQ ID NO: 13 Jag1-R-qRT5′-CCCCGTAGTGACAAGGGTTC-3′ SEQ ID NO: 14  8 Lif-F-qRT5′-AGCGCCAATGCTCTCTTCAT-3′ SEQ ID NO: 15 Lif-R-qRT5′-CAGTGGGGTTCAGGACCTTC-3′ SEQ ID NO: 16  9 Tnf-F-qRT5′-AGGCACTCCCCCAAAAGATG-3′ SEQ ID NO: 17 Tnf-R-qRT5′-CCACTTGGTGGTTTGTGAGTG-3′ SEQ ID NO: 18 10 Vegfa-F-qRT5′-CGATTGAGACCCTGGTGGAC-3′ SEQ ID NO: 19 Vegfa-R-qRT5′-GCTGGCTTTGGTGAGGTTTG-3′ SEQ ID NO: 20 11 Vegfb-F-qRT5′-GTGGTGCCATGGATAGACGTT-3′ SEQ ID NO: 21 Vegfb-R-qRT5′-ATGCTCCCGGGGTAGAGTC-3′ SEQ ID NO: 22 12 Fgf2-F-qRT5′-AAGCGGCTCTACTGCAAGAA-3′ SEQ ID NO: 23 Fgf2-R-qRT5′-TGTAACACACTTAGAAGCCAGCA-3′ SEQ ID NO: 24 13 GDNF-F-qRT5′-CAAAAATCGGGGGTGCGTTT-3′ SEQ ID NO: 25 GDNF-R-qRT5′-GCCTTCTACTCCGAGACAGG-3′ SEQ ID NO: 26 14 TGF-α-F-qRT5′-CCGGTTTTTGGTGCAGGAAG-3′ SEQ ID NO: 27 TGF-α-R-qRT5′-CACCACTCACAGTGTTTGCG-3′ SEQ ID NO: 28

All percentages and ratios are calculated by weight unless otherwiseindicated.

All percentages and ratios are calculated based on the total compositionunless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of treating Gaucher disease in anindividual in need thereof, comprising administering intravenously tosaid individual a composition comprising a Very Late Antigen-4 positiveneural precursor cell (“VLA4+NPC”) having a glucocerebrosidase (GCase)gene cloned into a lentiviral expression vector under the control of apromoter; wherein said GCase gene encodes for a functional GCase enzyme;wherein said VLA4+NPC cells are derived from an induced pluripotent stemcell (iPSC); and wherein said VLA4+NPC cells comprise at least 90% oftotal neural precursor cells (NPC) in said composition.
 2. The method ofclaim 1, wherein said Gaucher disease is in an individual having amutation in glucocerebrosidase gene GBA1.
 3. The method of claim 1,wherein said Gaucher disease is type II neuronopathic Gaucher diseasenGD.
 4. The method of claim 1, wherein said Gaucher disease is type IIIneuronopathic Gaucher disease nGD.
 5. The method of claim 1 wherein saidlentivirus comprises a human elongation factor 1 alpha (“EF1α”) promoteror an Ubiquitin C promoter (UbC).
 6. The method of claim 1 wherein saidVLA4+NPC is co-administered with a chaperone molecule.
 7. The method ofclaim 1 wherein said VLA4+NPC is co-administered with a chaperonemolecule selected from Dantrolene, Ambroxol, or a combination thereof.8. The method of claim 1 wherein said VLA4+NPC is delivered to saidindividual until one or more parameters selected from neurologicalpathology, survival, brain inflammation, brain neurodegeneration, GCaseactivity, GCase substrate level, mitochondrial function, neurotropicfactor expression, or combinations thereof, is improved.
 9. The methodof claim 1, wherein said VLA4+NPC is co-administered with an agentselected from imiglucerase, velaglucerase alfa, taliglucerase alpha,eligustat, miglustat, ibiglustat, or combinations thereof.